Biological compositions and methods for treatment of lymphoma

ABSTRACT

The present invention relates to pharmaceutical compositions and dietary supplement comprising yeast cells that can produce a healthful benefit in a subject inflicted with lymphoma. The biological compositions can be used to retard the growth of lymphoma cells and/or prolonging the time of survival of the subject. The invention also relates to methods for manufacturing the biological compositions.

1. FIELD OF THE INVENTION

[0001] The invention relates to oral compositions comprising yeast cellsthat can produce a healthful benefit in a subject inflicted withlymphoma. The invention also relates to methods for manufacturing theoral compositions and methods of use thereof.

2. BACKGROUND OF THE INVENTION 2.1 Lymphoma

[0002] Malignant lymphomas (ML) involve the cells of the lymphaticsystem, and is the fifth most common cancer in the U.S. ML includeHodgkin's disease, and non-Hodgkin's diseases which are a heterogeneousgroup of lymphoid proliferative diseases. Hodgkin's disease accounts forapproximately 14% of all malignant lymphomas. Incidence rates rise afterage 10, peak in late 20's, decline until age 45, and then increase againwith age.

[0003] Limited radiation therapy is recommended for early stages ofHodgkin's disease, while chemotherapy with or without radiation isreserved for advanced stages. Almost 70% of patients with Hodgkin'sdisease in the U.S. are long time survivors. Successful chemotherapeuticregimens use a combination of nitrogen mustard, vincristine, prednisone,and procarbazine, or doxorubicin, bleomycin, vinblastine, anddacarbazine. The incidence of non-Hodgkin's diseases rises inimmunosuppressed patients (including AIDS patients, renal transplantrecipients), patients with hyperfunctioning immune systems, andinfections by viruses, such as Epstein Barr virus (EBV), andT-lymphotropic virus type I infection. Non-Hodgkin's disease frequentlypresents as widely disseminated disease. The prime treatment modality ischemotherapy with radiation therapy used as an adjunct in the managementof a localized disease. Successful chemotherapeutic regimens use acombination of vincristine, prednisone, doxorubicin, bleomycin,vinblastine, methotrexate, dexamethasone and leucovorin.

[0004] The identification of active chemotherapeutic agents againstcancers traditionally involved the use of various animal models ofcancer. The mouse has been one of the most informative and productiveexperimental system for studying carcinogenesis (Sills et al., 2001,Toxicol Letters 120:187-198), cancer therapy (Malkinson, 2001, Lymphoma32(3):265-279; Hoffmnan R M., 1999, Invest New Drugs 17(4):343-359), andcancer chemoprevention (Yun, 1999, Annals N.Y. Acad Sci. 889:157-192).Cancer research started with transplanted tumors in animals whichprovided reproducible and controllable materials for investigation.Pieces of primary animal tumors, cell suspensions made from thesetumors, and immortal cell lines established from these tumor cellspropagate when transplanted to animals of the same species.

[0005] To transplant human cancer to an animal and to prevent itsdestruction by rejection, the immune system of the animal arecompromised. While originally accomplished by irradiation, thymectomy,and application of steroids to eliminate acquired immunity, nude micethat are athymic congenitally have been used as recipients of a varietyof human tumors (Rygaard, 1983, in 13^(th) International Cancer CongressPart C, Biology of Cancer (2), pp37-44, Alan R. Liss, Inc., N.Y.;Fergusson and Smith, 1987, Thorax, 42:753-758). While the athymic nudemouse model provides useful models to study a large number of humantumors in vivo, it does not develop spontaneous metastases and are notsuitable for all types of tumors. Next, the severe combinedimmunodeficient (SCID) mice is developed in which the acquired immunesystem is completely disabled by a genetic mutation. Human lymphoma wasfirst used to demonstrate the successful engraftment of a human cancerin the SCID mouse model (Reddy S., 1987, Cancer Res. 47(9):2456-2460).Subsequently, the SCID mouse model have been shown to allow disseminatedmetastatic growths for a number of human tumors, particularlyhematologic disorders and malignant melanoma (Mueller and Reisfeld,1991, Cancer Metastasis Rev. 10(3):193-200; Bankert et al., 2001, TrendsImmunol. 22:386-393). With the recent advent of transgenic technology,the mouse genome has become the primary mammalian genetic model for thestudy of cancer (Resor et al., 2001, Human Molec Genet. 10:669-675).

[0006] While surgery, chemotherapeutic agents and radiation are usefulin the treatment of lymphoma, there is a continued need to find bettertreatment modalities and approaches to manage the disease that are moreeffective and less toxic, especially when clinical oncologists aregiving increased attention to the quality of life of cancer patients.The present invention provides an alternative approach to cancer therapyand management of the disease by using an oral composition comprisingyeasts.

2.2 Yeast-Based Compositions

[0007] Yeasts and components thereof have been developed to be used asdietary supplement or pharmaceuticals. However, none of the priormethods uses yeast cells which have been cultured in an electromagneticfield to produce a product that has an anti-cancer effect. The followingare some examples of prior uses of yeast cells and components thereof:

[0008] U.S. Pat. No. 6,197,295 discloses a selenium-enriched dried yeastproduct which can be used as dietary supplement. The yeast strainSaccharomyces boulardii sequela PY 31 (ATCC 74366) is cultured in thepresence of selenium salts and contains 300 to about 6,000 ppmintracellular selenium. Methods for reducing tumor cell growth byadministration of the selenium yeast product in combination withchemotherapeutic agents is also disclosed.

[0009] U.S. Pat. No. 6,143,731 discloses a dietary additive containingwhole β-glucans derived from yeast, which when administered to animalsand humans, provide a source of fiber in the diet, a fecal bulkingagent, a source of short chain fatty acids, reduce cholesterol and LDL,and raises HDL levels.

[0010] U.S. Pat. No. 5,504,079 discloses a method of stimulating animmune response in a subject utilizing modified yeast glucans which haveenhanced immunobiologic activity. The modified glucans are prepared fromthe cell wall of Saccharomyces yeasts, and can be administered in avariety of routes including, for example, the oral, intravenous,subcutaneous, topical, and intranasal route.

[0011] U.S. Pat. No. 4,348,483 discloses a process for preparing achromium yeast product which has a high intracellular chromium content.The process comprises allowing the yeast cells to absorb chromium undera controlled acidic pH and, thereafter inducing the yeast cells to growby adding nutrients. The yeast cells are dried and used as a dietarysupplement.

[0012] Citation of documents herein is not intended as an admission thatany of the documents cited herein is pertinent prior art, or anadmission that the cited documents are considered material to thepatentability of the claims of the present application. All statementsas to the date or representations as to the contents of these documentsare based on the information available to the applicant and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

3. SUMMARY OF THE INVENTION

[0013] The present invention relates to biological or oral compositionsuseful for subjects with lymphoma. In one embodiment, the presentinvention provides biological compositions comprising live yeast cellswhich are capable of producing a healthful benefit in subjects withlymphoma. In other embodiments, the invention provides methods of makingthe biological compositions, and methods of using the biologicalcompositions.

[0014] In particular, the methods of the invention comprise culturingyeast cells in the presence of a series of electromagnetic fields suchthat the yeast cells becomes metabolically active. The electromagneticfields used are each defined by one of five frequency ranges and a broadrange of field strength. The starting yeast cells are commerciallyavailable and/or accessible to the public, such as but not limited toSaccharomyces. The methods for making the biological compositions of theinvention further comprise conditioning the activated yeast cells inplant extracts and the gastric juice of animals, while in the presenceof another series of electromagnetic fields.

[0015] The methods of manufacturing also comprise expanding the numberof activated or activated and conditioned yeast cells in large scalecultures in the presence of yet another series of electromagneticfields, performing quality control measures, and packaging.Pharmaceutical compositions of the invention comprises activated andconditioned yeast cells and one or more pharmaceutically acceptableexcipients or carriers. Additional ingredients, such as vitamins and/orflavors may be added to the biological compositions to form the oralcompositions of the invention. Such additional carriers and ingredientscan improve the healthful benefits, pharmacological properties, andorganoleptic characteristics of the oral compositions. During themanufacturing process, the activated or activated and conditioned yeastcells may be dried and stored for a period of time.

[0016] The biological or oral compositions of the invention are ingestedby the subject or used as an additive to be incorporated into food to beconsumed by the subject. Dietary supplement and nutritional compositionscomprising activated and conditioned yeast cells are encompassed by theinvention. Preferably, the subject is a human being.

[0017] In various embodiments, the biological or oral compositions ofthe invention are used to produce a healthful benefit in a subject withlymphoma or at high risk of developing lymphoma. In particular, thebiological composition of the invention can retard the growth oflymphoma cells in an animal which received the composition orally. Thecomposition can also be used to prolong the time of survival of ananimal with lymphoma.

4. BRIEF DESCRIPTION OF FIGURES

[0018]FIG. 1 Activation and conditioning of yeast cells. 1 yeast cellculture; 2 container; 3 electromagnetic field source; 4 electrode.

[0019]FIG. 2 Large scale propagation of yeast cells. 5 first container;6 second container; 7 third container; 8 yeast cell cultures; 9electromagnetic field source.

5. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to biological compositions that canproduce a healthful benefit in a subject with lymphoma. The presentinvention provides methods for manufacturing the biological compositionsas well as methods for using the biological compositions.

[0021] In one embodiment, the invention provides biological compositionsthat comprise yeasts. Unlike the traditional use of yeasts in the makingof food, the yeast cells of the invention are not used as a source ofenzymes that acts on the food ingredients. The yeasts are not a primarysource of nutrients for the subject. Nor are yeast cells used as acarrier, such as metal salts. The yeast cells of the invention are livewhen administered orally or ingested along with food by a subject.Without being bound by any theory or mechanism, the inventor believesthat the culture conditions activate and/or amplified the expression ofa gene or a set of genes in the yeast cells such that the yeast cellsbecomes highly effective in stimulating the animal's immune system,including both specific and non-specific immunological reactions, theresults of which are manifested as the overall healthful benefitsobserved in the treated subject. The healthful benefits provided byusing the biological compositions are demonstrated in animal models ofhuman lymphoma which show inhibition of tumor growth and prolongedsurvival time of animals with the disease.

[0022] In another embodiment, the invention provides methods for makingthe yeast cells in the biological compositions. The starting materialsare normal yeast cells which can be readily obtained commercially orfrom public microorganism deposits. The methods of the inventioncomprise a set of culture conditions that can be applied reproducibly toactivate the yeast cells. The key feature of the culture conditions usedin the methods of the invention is a series of alternatingelectromagnetic fields of defined frequency ranges and field strengthswhich are applied to the growing yeast cell culture. The method furthercomprises the step of conditioning the activated live yeast cells to theacidic environment of the stomach of the subject. The electromagneticfields used in these methods can be created reproducibly at variousscales, thus enabling even the large scale manufacturing of thebiological compositions of the invention. By careful control of theculturing conditions, normal yeast cells can be activated routinely andreproducibly to become yeast cells of the invention.

[0023] In yet another embodiment, the invention provides methods formanufacturing an oral composition comprising activated and conditionedyeasts of the invention, and additional ingredients, including but notlimited to pharmaceutically acceptable carriers or excipients, vitamins,herbs (including traditional Chinese medicine products), herbalextracts, minerals, amino acids, flavoring agents, coloring agents,and/or preservatives.

[0024] In yet another embodiment, the biological compositions can beadded to food which will be consumed by the subject. As known to thoseskilled in the relevant art, many methods may be used to mix thebiological or oral compositions of the invention with food while theyeast cells remain viable. In a particular embodiment, the culture brothcomprising live yeast cells of the present invention are added directlyto food just prior to consumption. Dried powders of the yeasts can alsobe reconstituted and added directly to food just prior to consumption.

[0025] In various embodiments, the oral compositions of the inventioncan be consumed directly by a subject or be fed directly to a subject.For example, the subject may drink the culture broth or a fractionthereof that comprises live activated and conditioned yeast cells. Oralcompositions comprising dried yeast cells can also be given as a soliddosage form to the subject.

[0026] Although it is not necessary, the biological or oral compositionsof the invention can be used in conjunction or in rotation with othertypes of treatment modalities such as but not limited to surgery,chemotherapeutic agents, and radiation. Since the biologicalcompositions of the invention are administered orally, the assistance ofhealth professionals in administration of the composition is generallynot essential.

[0027] Described below in Section 5.1 are the yeast cells of theinvention and methods of their preparation. Section 5.2 describes theuse of the biological compositions of the invention in a subjectsuffering from lymphoma. The examples in Sections 6 to 9 demonstrate thetherapeutic benefits of an oral composition of the invention. Theactivated and conditioned yeast cells in the oral composition arecharacterized by their ability to (i) suppress the growth of cancercells in an animal model of human lymphoma, or (ii) prolong the survivalof animals with transplanted cancer cells in a model of human lymphoma,as compared to yeast cells which have not been activated andconditioned.

5.1 Preparation of the Yeast Cell Cultures

[0028] The yeast cells of the biological composition are produced byculturing a plurality of yeast cells in an appropriate culture medium inthe presence of an alternating electromagnetic field over a period oftime. The method comprises a first step of activating the yeast cellsand a second step of conditioning the activated yeast cells. Theactivation process comprises culturing yeast cells in the presence of atleast two, three, four or five electromagnetic fields of specificfrequencies and field strength. The conditioning process comprisesfurther culturing of the activated yeast cells in a medium comprisingplant extracts and extracts from the stomach of an animal, in thepresence of at least one electromagnetic field. The activated andconditioned yeast cells can be stored as dried cells after drying thecells under appropriate conditions. The dried activated and conditionedyeast cells can be used later in large scale culturing processes formanufacturing the biological compositions of the invention. The variousculturing processes of the invention can be performed either as a batchprocess or a continuous process.

5.1.1 Yeasts

[0029] In various embodiments, yeasts of the genera of Saccharomyces,Candida, Crebrothecium, Geotrichum, Hansenula, Kloeckera, Lipomyces,Pichia, Rhodosporidium, Rhodotorula, Torulopsis, Trichosporon, andWickerhamia can be used in the invention. Generally, fungi used for foodmanufacturing are preferred.

[0030] Non-limiting examples of yeast strains include Saccharomyces sp.,AS2.311; Schizosaccharomyces pombe Linder, AS2.214, AS2.248, AS2.249,AS2.255, AS2.257, AS2.259, AS2.260, AS2.274, AS2.994, AS2.1043,AS2.1149, AS2.1178, IFFI 1056; Saccharomyces sake Yabe, ACCC2045;Saccharomyces uvarum Beijer, IFFI 1023, IFFI 1032, IFFI 1036, IFFI 1044,IFFI 1072, IFFI 1205, IFFI 1207; Saccharomyces rouxii Boutroux, AS2.178,AS2.180, AS2.370, AS2.371; Saccharomyces cerevisiae Hansen Var.ellipsoideus, ACCC2043, AS2.2, AS2.3, AS2.8, AS2.53, AS2.163, AS2.168,AS2.483, AS2.541, AS2.559, AS2.606, AS2.607, AS2.611, AS2.612;Saccharomyces carlsbergensis Hansen, AS2.116, AS2.162, AS2.189, AS2.200,AS2.216, AS2.265, AS2.377, AS2.417, AS2.420, AS2.440, AS2.441, AS2.443,AS2.444, AS2.459, AS2.595, AS2.605, AS2.638, AS2.742, AS2.745, AS2.748,AS2.1042; Rhodotorula aurantiaca (Saito) Ladder; AS2.102, AS2.107,AS2.278, AS2.499, AS2.694, AS2.703, AS2.704 and AS2.1146; Saccharomycescerevisiae Hansen, ACCC2034, ACCC2035, ACCC2036, ACCC2037, ACCC2038,ACCC2039, ACCC2040, ACCC2041, ACCC2042, AS2.1, AS2.4, AS2.11, AS2.14,AS2.16, AS2.56, AS2.69, AS2.70, AS2.93, AS2.98, AS2.101, AS2.109,AS2.110, AS2.112, AS2.139, AS2.173, AS2.182, AS2.196, AS2.242, AS2.336,AS2.346, AS2.369, AS2.374, AS2.375, AS2.379, AS2.380, AS2.382, AS2.393,AS2.395, AS2.396, AS2.397, AS2.398, AS2.399, AS2.400, AS2.406, AS2.408,AS2.409, AS2.413, AS2.414, AS2.415, AS2.416, AS2.422, AS2.423, AS2.430,AS2.431, AS2.432, AS2.451, AS2.452, AS2.453, AS2.458, AS2.460, AS2.463,AS2.467, AS2.486, AS2.501, AS2.502, AS2.503, AS2.504, AS2.516, AS2.535,AS2.536, AS2.558, AS2.560, AS2.561, AS2.562, AS2.576, AS2.593, AS2.594,AS2.614, AS2.620, AS2.628, AS2.631, AS2.666, AS2.982, AS2.1190,AS2.1364, AS2.1396, IFFI 1001, IFFI 1002, IFFI 1005, IFFI 1006, IFFI1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI 1021, IFFI 1027, IFFI 1037,IFFI 1042, IFFI 1045, IFFI 1048, IFFI 1049, IFFI 1050, IFFI 1052, IFFI1059, IFFI 1060, IFFI 1062, IFFI 1202, IFFI 1203, IFFI 1209, IFFI 1210,IFFI 1211, IFFI 1212, IFFI 1213, IFFI 1215, IFFI 1221, IFFI 1224, IFFI1247, IFFI 1248, IFFI 1251, IFFI 1270, IFFI 1277, IFFI 1289, IFFI 1290,IFFI 1291, IFFI 1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 1301, IFFI1302, IFFI 1307, IFFI 1308, IFFI 1309, IFFI 1310, IFFI 1311, IFFI 1331,IFFI 1335, IFFI 1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI 1340, IFFI1345, IFFI 1348, IFFI 1396, IFFI 1397, IFFI 1399, IFFI 1441 and IFFI1443. Preferred yeast strains include but are not limited to S.cerevisiae AS2.501, AS2.502, AS2.503, AS2.504, AS2.535, AS2.558,AS2.560, AS2.561 and AS2.562.

[0031] Generally, yeast strains useful for the invention can be obtainedfrom private or public laboratory cultures, or publicly accessibleculture deposits, such as the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209 and the China GeneralMicrobiological Culture Collection Center (CGMCC), China Committee forCulture Collection of Microorganisms, Institute of Microbiology, ChineseAcademy of Sciences, Haidian, P.O. Box 2714, Beijing, 100080, China.

[0032] Non-limiting examples of using yeast cells of the invention withSaccharomyces cerevisiae Hansen strain AS2.562 are provided in Sections6 to 9 herein below. The yeast cells of the invention do not comprise anenhanced level of selenium or chromium relative to that found innaturally occurring yeast cells. In certain embodiments, the biologicalcompositions do not comprise cells of Saccharomyces boulardii (forexample, ATCC Accession No. 74366) or cells of a particular strain ofSaccharomyces cerevisiae (strain Hansen CBS 5926) that is also commonlyreferred to as Saccharomyces boulardii.

[0033] Although it is preferred, the preparation of the yeast cells ofthe invention is not limited to starting with a pure strain of yeast.The yeast cells in the biological compositions may be produced byculturing a mixture of yeast cells of different species or strains. Theconstituents of a mixture of yeast cells can be determined by standardyeast identification techniques well known in the art.

[0034] In various embodiments of the invention, standard techniques forhandling, transferring and storing yeasts are used. Although it is notnecessary, sterile conditions or clean environments are highly desirablewhen carrying out the manufacturing processes of the invention,especially when the biological compositions are for human consumption.The manufacturing process can be adapted to meet regulatory guidelineson product safety and quality control by standard practice known in theart.

5.1.2 Electromagnetic Fields

[0035] As used herein, the terms “alternating electromagnetic field”,“electromagnetic field” or “EM field” are synonymous. An electromagneticfield useful in the invention can be generated by various means wellknown in the art. A schematic illustration of exemplary setups aredepicted respectively in FIG. 1. An electromagnetic field of a desiredfrequency and a desired field strength is generated by anelectromagnetic wave source (3) which comprises one or more signalgenerators that are capable of generating electromagnetic waves,preferably sinusoidal waves, and preferably in the frequency range of1,500 to 15,000 MHz and most preferably 7,900 to 12,900 MHz. Such signalgenerators are well known in the art. Signal generators capable ofgenerating signal with a narrower frequency range can also be used. Ifdesirable, a signal amplifier can also be used to increase the outputsignal, and thus the strength of the EM field.

[0036] The electromagnetic field can be applied to the culture by avariety of means including placing the yeast cells in close proximity toa signal emitter connected to a source of electromagnetic waves. Thesignal generator is connected to the signal emitter by cables such ascoaxial cables that can transmit signals up to greater than or equal to30 GHz. Typically, the yeast cells are placed in a container which ismade of material that is not an electric conductor, such as but notlimited to plastic, resin, glass, and ceramic.

[0037] In one embodiment, the electromagnetic field is applied by signalemitters in the form of electrodes (4) that are submerged in a cultureof yeast cells (1). In a preferred embodiment, one of the electrodes isa metal plate which is placed on the bottom of a non-conductingcontainer (2), and the other electrode comprises a plurality of wires ortubes so configured inside the container such that the energy of theelectromagnetic field can be evenly distributed in the culture. Theelectrodes are preferably made of copper. For an upright culture vessel,the tips of the wires or tubes are placed within 3 to 30 cm from thebottom of the vessel (i.e., approximately 2% to 10% of the height of thevessel from the bottom). Table 1 provides exemplary set up for culturingthe yeast cells of the invention. TABLE 1 height of culture medium indistance electrodes are range for distance of the non-conducting placedfrom the bottom the electrodes from the container (cm) of the container(cm) bottom (cm) 15 to 20 3 3 to 5 20 to 30 5 5 to 7 30 to 50 7  7 to 1050 to 70 10 10 to 15  70 to 100 15 15 to 20 100 to 150 20 20 to 30 150to 200 30 25 to 30

[0038] The number of electrodes used depends on both the volume of theculture and the diameter of the electrode. For example, for a culturehaving a volume of 10 liter or less, two or three electrodes having adiameter of between 0.5 to 2.0 mm can be used. For a culture volume of10 to 100 liter of culture, the electrodes can have a diameter of 3.0 to5.0 mm. For a culture volume of 100 to 1,000 liter, the electrodes canhave a diameter of 6.0 to 15.0 mm. For a culture having a volume greaterthan 1,000 liter, the electrodes can have a diameter of between 20.0 to25.0 mm.

5.1.3 Activation of Yeast Cells

[0039] According to the invention, the method for producing activatedyeast cells of the invention comprises culturing yeast cells in thepresence of at least two, three, four or five alternatingelectromagnetic (EM) fields.

[0040] The culture process can be initiated by inoculating 1,000 ml ofmedium with an inoculum of a selected yeast strain (such as one of thosedescribed in Section 5.1.1) such that the starting cell density of theculture is greater than about 10⁵ cells per ml. For example,Saccharomyces cerevisiae Hansen strain AS2.562 can be used. The startingculture can be used to seed larger scale culture. The culture ismaintained initially at 28° C. to 32° C. for 22 to 30 hours prior toexposure to the EM field(s), typically at 30° C. for 28 hours.

[0041] The culturing process may preferably be conducted underconditions in which the concentration of dissolved oxygen is between0.025 to 0.08 mol/m³, preferably 0.04 mol/m³. The oxygen level can becontrolled by any conventional means known in the art, including but notlimited to stirring and/or bubbling.

[0042] The culture is most preferably carried out in a liquid mediumwhich contains sources of nutrients assimilable by the yeast cells.Table 2 provides an exemplary medium for culturing the yeast cells ofthe invention. TABLE 2 Medium Composition Quantity Sucrose or glucose 20g Vitamin H 60 μg Vitamin B₆ 40 μg Vitamin B₁₂ 40 μg KH₂PO₄ 0.20 gMgSO₄.7H₂O 0.20 g NaCl 0.20 g CaSO₄.2H₂O 0.20 g CaCO₃.5H₂O 3.0 g Peptone2.5 g Autoclaved water 1,000 ml

[0043] The culturing medium is heated to 45° C. and cooled before addingthe vitamin H, vitamin B₆, and vitamin B₁₂.

[0044] In general, carbohydrates such as sugars, for example, sucrose,glucose, fructose, dextrose, maltose, xylose, and the like and starches,can be used either alone or in combination as sources of assimilablecarbon in the culture medium. The exact quantity of the carbohydratesource or sources utilized in the medium depends in part upon the otheringredients of the medium but, in general, the amount of carbohydrateusually varies between about 0.1% and 5% by weight of the medium and ispreferably between about 0.2% and 2%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium. Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are KH₂PO₄, (NH₄)₂HPO₄, CaCO₃,MgSO₄, NaCl, and CaSO₄.

[0045] It should be noted that the composition of the media provided inTable 2 is not intended to be limiting. The process can be scaled up ordown according to needs. Various modifications of the culture medium maybe made by those skilled in the art, in view of practical and economicconsiderations, such as the scale of culture and local supply of mediacomponents.

[0046] In certain embodiments, a series of at least two, three, four orfive EM fields are applied to the culture of yeast cells, each having adifferent frequency within a stated range, and a different fieldstrength within a stated range. The EM fields can be applied in anyorder and by any means known in the art, such as the apparatus describedin Section 5.1.2. Although any of the following two, three or four EMfields can be applied, preferably, all five EM fields are applied.

[0047] For the first EM field, the frequency is in the range of 7,972 to2,983 MHz and the field strength is in the range of 250 to 300 mV/cm.The yeast culture is exposed to this first EM field at 30±2° C. forabout 18 to 22 hours, preferably 20 hours.

[0048] For the second EM field, the frequency is in the range of 7,993to 8,003 MHz and the field strength is in the range of 250 to 300 mV/cm.The yeast culture is exposed to this second EM field at 30±2° C. forabout 18 to 22 hours, preferably 20 hours.

[0049] For the third EM field, the frequency is in the range of 10,024to 10,034 MHz and the field strength is in the range of 370 to 400mV/cm. The yeast culture is exposed to this third EM field at 30±2° C.for about 20 to 25 hours, preferably 22 hours.

[0050] For the fourth EM field, the frequency is in the range of 12,832to 12,842 MHz and the field strength is in the range of 420 to 450mV/cm. The yeast culture is exposed to this fourth EM field at 30±2° C.for about 16 to 22 hours, preferably 22 hours.

[0051] For the fifth EM field, the frequency is in the range of 12,857to 12,867 MHz and the field strength is in the range of 310 to 350mV/cm. The yeast culture is exposed to this fifth EM field at 30±2° C.for about 10 to 15 hours, preferably 12 hours.

[0052] In less preferred embodiments, the yeast cells can be cultured byexposure to two, three or four of the above-mentioned EM fields in adifferent order. The yeast cells can remain in the same container anduse the same set of electromagnetic wave generator and emitters whenswitching from one EM field to another EM field.

[0053] The cell density of the culture at the end of the activationprocess is typically greater than about 10⁶ to 10⁹ cells per ml(estimated by hematocytometer). The activated yeast cells may berecovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C. The activated yeastcells recovered from the liquid culture may be dried and stored inpowder form. Preferably, the powder form of the yeast cells comprisesgreater than about 10⁷ to 10¹⁰ yeast cells per gram.

5.1.4 Conditioning of Yeast Cells

[0054] According to the invention, performance of the activated yeastcells can be optimized by culturing the activated yeast cells in thepresence of an extract from the stomach (e.g., the gastric juice) of ananimal with physiology similar to the subject to which the biologicalcomposition will be administered. The inclusion of this additionalconditioning process allows the activated yeast cells to adapt to andendure the acidic environment of the subject's stomach. The method forconditioning activated yeast cells of the invention comprises culturingyeast cells in such materials in the presence of at least one EM field.

[0055] The culture process can be initiated by inoculating 1,000 ml of aconditioning medium with about 10 gram of dried activated yeastscontaining about 10¹⁰ cells per gram (as prepared by the methodsdescribed in Section 5.1.3). An equivalent number of yeast cells inculture, preferably greater than 10⁶ to 10⁹ cells per ml, morepreferably at 10⁸ cells per ml, can also be used as an inoculum. Theconditioning medium comprises per 1,000 ml about 700 ml of gastric juiceof an animal and about 300 ml of wild hawthorn juice. The process can bescaled up or down according to needs.

[0056] The gastric juice of an animal can be obtained from the stomachcontent of a freshly slaughtered animal. Although not essential, theanimal is preferably kept under a clean environment, and fed a standarddiet, preferably germ-free. For example, the content of the stomach of a120-day old pig is mixed with 2,000 ml of distilled water, and allowedto settle without stirring for 6 hours. The clear liquid above iscollected for use as the gastric juice used in the conditioning process.The gastric juice of a pig can be used to condition yeast cells for usein a variety of mammals, including humans. Other methods that can beused to collect the gastric juice include centrifugation or filtrationof the mixture to remove debris and/or microorganisms. The gastric juiceso obtained can be stored at 4° C. Preferably, the collection proceduresand storage are carried out under sterile conditions.

[0057] The wild hawthorn juice is an extract of wild hawthorn fruitsprepared by slicing the fruits and drying the slices in air, preferablyto less than 8% moisture (commercial dryer can be used if necessary),crushing the dried fruits to less than 20 mesh, and mixing 1,500 ml ofwater per 500 gram of the crushed wild hawthorn. The mixture is thenallowed to settle without stirring for 6 hours, and the clear liquidabove is collected for use as the wild hawthorn juice used in theconditioning process. Other methods that can be used to collect thehawthorn juice include centrifugation or filtration of the mixture.Preferably, the collection procedures and storage are carried out understerile conditions.

[0058] The activated yeast cells are conditioned by culturing in atleast one of the following two EM fields which can be applied by theapparatus described in Section 5.1.2 or any means known in the art:

[0059] The first EM field has a frequency in the range of 12,832 to12,842 MHz and a field strength in the range of 380 to 400 mV/cm. Thetemperature is maintained at 28° C. to 32° C., and typically at 30° C.The yeast culture is exposed to this first EM field for about 36 to 42hours, preferably 39 hours.

[0060] The second EM field has a frequency in the range of 12,857 to12,867 MHz and a field strength in the range of 300 to 350 mV/cm. Thetemperature is maintained at 28° C. to 32° C., and typically at 30° C.The yeast culture is exposed to this second EM field for about 18 to 25hours, preferably 20 hours.

[0061] In a preferred embodiment, the activated yeast cells areconditioned by culturing in both of the above-mentioned EM fields. Inless preferred embodiments, the yeast cells are conditioned in the twodifferent EM fields in a different order. In other embodiments, a seriesof EM fields having field characteristics within the ranges stated abovecan be applied to condition the yeast cells. The yeast cells can remainin the same container and use the same set of electromagnetic wavegenerator and emitters when switching from one EM field to another EMfield.

[0062] The cell density of the culture at the end of the activationprocess is typically greater than about 10⁷ to 10¹⁰ cells per ml(estimated by hematocytometer). The activated and conditioned yeastcells may be recovered from the culture by various methods known in theart, and stored at a temperature below about 0° C. to 4° C.

[0063] The activated and conditioned yeast cells can be used directly ina biological composition or used as a starter culture for large scalemanufacturing. The activated and conditioned yeast cells recovered fromthe liquid culture may be dried and stored in powder form. Preferably,the powder form of the activated and conditioned yeast cells comprisesgreater than about 10⁸ to 10¹¹ yeast cells per gram.

5.1.5 Large Scale Manufacturing

[0064] The present invention also encompasses methods of manufacturingof the biological compositions of the invention at a large scale. Theactivated and conditioned yeast cells as prepared by Sections 5.1.3 and5.1.4 are propagated on a large scale to make the biologicalcompositions of the invention. The method comprises culturing the yeastcells in the presence of one or more EM fields for a period of time,diluting the growing yeast cells with fresh medium, and repeating theprocess. The method can be carried out as a batch process or acontinuous process.

[0065] In one preferred embodiment, a set of three containers (5, 6, 7)each comprising a set of electrodes for generating an electromagneticfield as described in Section 5.1.2 are set up each with 1,000 liters ofa culture medium. See FIG. 2. The culture medium comprises nutrientsassimilable by the yeast cells as shown in Table 3. TABLE 3 MaterialQuantity Wild hawthorn juice 300 liters Jujube juice 300 liters Wu weizi juice 300 liters Soybean juice 100 liters

[0066] The wild hawthorn juice is an extract of fresh wild hawthornfruits prepared by washing the fruits clean, drying the fruits in air orusing a commercial dryer to less than 8% moisture, crushing the driedfruits to less than 20 mesh, and mixing the crushed wild hawthorn withwater at a ratio of 400 liters of water per 100 kg of crushed fruits.The mixture is then stirred continuously for 12 hours while thetemperature is maintained at 28° C. to 30° C. The mixture is thencentrifuged at 1,000 rpm to collect the supernatant which is used asdescribed above. Preferably, the procedures are carried out understerile conditions.

[0067] The jujube juice is an extract of fresh jujube fruits prepared bywashing the fruits clean, drying the fruits to less than 8% moisture,crushing the dried fruits to less than 20 mesh, and mixing the crushedjujube with water at a ratio of 400 liters of water per 100 kg ofcrushed fruits. The mixture is then stirred continuously for 12 hourswhile the temperature is maintained at 28° C. to 30° C. The mixture isthen centrifuged at 1,000 rpm to collect the supernatant which is usedas described above. Preferably, the procedures are carried out understerile conditions.

[0068] The wu wei zi juice is an extract of fresh berries of Schisandrachinensis plant prepared by washing the berries, drying the fruits toless than 8% moisture, crushing the dried berries to less than 20 mesh,and mixing the crushed berries with water at a ratio of 400 liters ofwater per 100 kg of crushed berries. The mixture is then stirredcontinuously for 12 hours while the temperature is maintained at 28° C.to 30° C. The mixture is then centrifuged at 1,000 rpm to collect thesupernatant which is used as described above. Preferably, the proceduresare carried out under sterile conditions.

[0069] The soybean juice is prepared by washing the soybeans, drying thesoybeans to less than 8% moisture, crushing the soybeans to less than 20mesh, and mixing the crushed soybeans with water. For 30 kg of soybeans,130 liters of water is used. The mixture is then stirred continuouslyfor 12 hours while the temperature is maintained at 28° C. to 30° C. Themixture is then centrifuged at 1,000 rpm to collect the supernatantwhich is used as described above. Preferably, the procedures are carriedout under sterile conditions.

[0070] The first container is inoculated with activated or activated andconditioned yeast cells as prepared by the methods of Sections 5.1.4 and5.1.5. About 1,000 gram of dried yeast powder are added to 1,000 literof culture medium. Each gram of the dried yeast powder comprises about10¹⁰ yeast cells. Instead of dried yeast cells, an equivalent number ofyeast cells in a liquid medium can also be used, preferably greater thanabout 10⁶ to 10⁹ cells per ml, more preferably about 10⁷ cells per ml.

[0071] The yeast cells in the first container (5) are then subjected toa series of two EM fields. For the first EM field, which can be appliedby the apparatus described in Section 5.1.2, the frequency is in therange of 12,832 to 12,842 MHz and the field strength is in the range of420 to 450 mV/cm. The yeast culture is exposed to this first EM fieldfor about 12 hours. The yeast cells are then subjected to a second EMfield having a frequency in the range of 12,857 to 12,867 MHz and afield strength in the range of 340 to 380 mV/cm. The yeast culture isexposed to this second EM field for about 10 hours. The yeast cells fromthe first container are then transferred to the second container whichcontains about 1,000 liter of the culture medium. In effect, the firstyeast culture is diluted by about 50% with fresh culture medium.

[0072] In the second container (6), the yeast cells are again subjectedto a series of two EM fields. The frequencies used in the secondcontainer are similar to those used in the first container but the fieldstrengths are marginally lower. The first EM field has a frequency inthe range of 12,832 to 12,842 MHz and a field strength in the range of330 to 350 mV/cm. The yeast culture is exposed to this EM field forabout 14 hours. The yeast cells are then subjected to a second EM fieldhaving a frequency in the range of 12,857 to 12,867 MHz and a fieldstrength in the range of 210 to 240 mV/cm. The yeast culture is exposedto this second EM field for about 10 hours. The yeast cells from thesecond container are then transferred to the third container whichcontains yet another 1,000 liter of the culture medium. Again, thesecond yeast culture is diluted by about 50% with fresh culture medium.

[0073] In the third container (7), the yeast cells are again subjectedto a series of two EM fields. The frequencies used in the thirdcontainer are similar to those used in the first and second containerbut the field strengths are lower. The first EM field has a frequency inthe range of 12,832 to 12,842 MHz and field strength in the range of 230to 250 mV/cm. The yeast culture is exposed to this EM field for about 22hours. The yeast cells are then subjected to a second EM field having afrequency in the range of 12,857 to 12,867 MHz and a field strength inthe range of 120 to 150 mV/cm. The yeast culture is exposed to this EMfield for about 12 hours.

[0074] The yeast cell culture resulting from the end of this stage canbe used directly as an oral composition of the invention, or used toform other compositions encompassed by the invention.

[0075] The cell density of the culture at the end of the large scalemanufacturing process is typically greater than about 10⁸ to 10¹⁰ cellsper ml (estimated by hematocytometer). The concentration of yeast cellsin the medium can be concentrated or diluted accordingly. In certainembodiments, the concentration of yeast cells in the medium is in therange of 10³ to 10¹⁰ cells per ml. In less preferred embodiments, theconcentration of yeast cells in the medium is in the range of 10³ to 10⁶cells per ml. In more preferred embodiments, the concentration of yeastcells in the medium is greater than 10⁶ to 10¹⁰ cells per ml. In mostpreferred embodiments, the concentration of yeast cells in the medium isin the range of 10⁶ to 5×10⁸ cells per ml.

[0076] Other ingredients that enhance the healthful benefits,pharmacological properties and/or organoleptic characteristics of thecomposition can be added to the yeast cell culture. To maintainviability and freshness of the composition, it is preferred that thevarious downstream and packaging process be carried out below roomtemperature, and preferably at 0° C. to 4° C. In one embodiment, theyeast cell culture can be packaged in liquid containers.

[0077] In another embodiment, the activated and conditioned yeast cellscan be dried as follows. The yeast cell culture is first centrifugedunder 75 to 100 g for 10 to 20 minutes to remove the supernatant. Theresidue which may contain up to 85% moisture is dried in a first dryerat a temperature not exceeding 60±2° C. for a period of 5 minutes sothat yeast cells quickly became dormant. The yeast cells were then sentto a second dryer and dried at a temperature not exceeding 65±2° C. fora period of about 8 minutes to further remove at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least99% of water. For example, the yeast cells may be dried to remove atleast 88% of water so the dried yeast cells may contain up to 12%moisture.

[0078] After cooling to room temperature, the dried yeast cells can bepackaged by standard pharmaceutical methods in various solid dosageform, each containing a predetermined amount of the dried material. In apreferred embodiment, the dried material comprises about 10⁵ to 10¹¹cells per gram. In a more preferred embodiment, the dried materialcomprises about 10⁸ to 5×10¹⁰ cells per gram. In a most preferredembodiment, the dried material comprises about 5×10⁸ cells per gram.

[0079] In general, the compositions are prepared by uniformly andintimately admixing the active ingredient with liquid carriers or finelydivided solid carriers.

5.1.6 PREFERRED EMBODIMENTS

[0080] In one preferred embodiment, the invention provides a method forpreparing a biological composition comprising activated and conditionedyeast cells, said method comprising in any order the steps of:

[0081] (a) culturing the yeast cells in a first electromagnetic fieldhaving a frequency at 7,977 MHz and a field strength of 282 mV/cm;

[0082] (b) culturing the yeast cells in a second electromagnetic fieldhaving a frequency at 7,998 MHz and a field strength of 277 mV/cm;

[0083] (c) culturing the yeast cells in a third electromagnetic fieldhaving a frequency at 10,029 MHz and a field strength of 387 mV/cm;

[0084] (d) culturing the yeast cells in a fourth electromagnetic fieldhaving a frequency at 12,837 MHz and a field strength of 442 mV/cm; and

[0085] (e) culturing the yeast cells in a fifth electromagnetic fieldhaving a frequency at 12,862 MHz and a field strength of 325 mV/cm;

[0086] and after the last of the first five steps, the following stepsin any order:

[0087] (f) culturing the yeast cells in a liquid medium comprising wildhawthorn juice and gastric juice of a mammal in a sixth electromagneticfield having a frequency at 12,837 MHz and a field strength of 391mV/cm; and

[0088] (g) culturing the yeast cells in a liquid medium comprising wildhawthorn juice and gastric juice of a mammal in a seventhelectromagnetic field having a frequency at 12,862 MHz and a fieldstrength of 315 mV/cm.

[0089] The activated and conditioned yeast cells obtained at theconclusion of this method is encompassed by the invention. Preferably,the yeast cells are Saccharomyces cerevisiae Hansen strain AS2.562.These yeast cells can be used in the following method of furtherexpanding number of activated and conditioned yeast cells.

[0090] In another preferred embodiment, the invention provides a methodof mass producing a biological composition comprising activated andconditioned yeast cells, said method comprising culturing the activatedand conditioned yeast cells prepared by the preferred embodimentdescribed above in this section, in a medium comprising wild hawthornjuice, jujube juice, wu wei zi juice, and soybean juice, and in thepresence of one or more series of electromagnetic fields. Each series ofEM fields comprises two EM fields in the order stated:

[0091] (h) an eighth electromagnetic field or series of electromagneticfields having a frequency at 12,837 MHz and a field strength in therange of 230 to 450 mV/cm, preferably at three fields strengths, e.g.,in the order of 442 mV/cm, 341 mV/cm, and 244 mV/cm; and

[0092] (i) a ninth electromagnetic field or series of electromagneticfields having a frequency at 12,862 MHz and a field strength in therange of 120 to 380 mV/cm, preferably at three fields strengths, e.g.,in the order of 350 mV/cm, 224 mV/cm, and 137 mV/cm.

[0093] The series may be repeated several times, such as three times,each time using a slightly lower field strength.

5.2 Methods of Uses 5.2.1 Uses in Subjects with Lymphoma

[0094] The present invention further provides methods of use of thebiological compositions of the invention. In one embodiment, thebiological composition is used as a medicament for treatment oflymphoma. In another embodiment, the biological composition is used as adietary supplement, health food, or health drink. The methods compriseadministering an effective amount of the biological composition to asubject in need. The biological composition may be administered orally,in liquid or solid form, or enterally through a feeding tube. As usedherein, the term “an effective amount” means an amount sufficient toprovide a therapeutic or healthful benefit in the context of lymphoma.

[0095] According to the invention, the biological composition canproduce a healthful benefit in a subject suffering from lymphoma.Preferably, the subject is a human being. The subject in need is one whois diagnosed with lymphoma, with or without metastasis, at any stage ofthe disease (e.g., I to IV, Ann Arbor Staging System). As used herein,the term “lymphoma” includes but is not limited to Hodgkin's disease andnon-Hodgkin's disease. For a detailed description of each type oflymphoma, see Rosenberg S A, Kaplan H S, eds. “Malignant Lymphomas:Etiology, Immunology, Pathology, Treatment” New York, N.Y., AcademicPress 1982, which is incorporated herein by reference in its entirety.Classic Hodgkin's disease is divided into four subtypes: (1) nodularsclerosis Hodgkin's disease (NSHD); (2) mixed cellularity Hodgkin'sdisease (MCHD); (3) lymphocyte depletion Hodgkin's disease (LDHD); and(4) lymphocyte-rich classic Hodgkin's disease (cLRHD). Non-Hodgkin'slymphoma includes but is not limited to (1) slow-growing lymphomas andlymphoid leukemia (e.g., chronic lymphocytic leukemia, small lymphocyticleukemia, lymphoplasmacytoid lymphoma, follicle center lymphoma,follicular small cleaved cell, follicular mixed cell, marginal zoneB-cell lymphoma, hairy cell leukemia, plasmacytoma, myeloma, largegranular lymphocyte leukemia, mycosis fungoides, sézary syndrome); (2)moderately aggressive lymphomas and lymphoid leukemia (e.g.,prolymphocytic leukemia, mantle cell lymphoma, follicle center lymphoma,follicular small cleaved cell, follicle center lymphoma, chroniclymphocytic leukemia/prolymphocytic leukemia, angiocentric lymphoma,angioimmunoblastic lymphoma); (3) aggressive lymphomas (e.g., largeB-cell lymphoma, peripheral T-cell lymphomas, intestinal T-celllymphoma, anaplastic large cell lymphoma); and (4) highly aggressivelymphomas and lymphoid leukemia (e.g., B-cell precursor B-lymphoblasticleukemia/lymphoma, Burkitt's lymphoma, high-grade B-cell lymphoma,Burkitt's-like T-cell precursor T-lymphoblastic leukemia/lymphoma).

[0096] The subject may be a lymphoma patient who is receivingconcurrently other treatment modalities against the lymphoma. Thesubject can be a lymphoma patient who had undergone a regimen oftreatment (e.g., chemotherapy and/or radiation) and whose cancer isregressing. The subject may be a lymphoma patient who had undergone aregimen of treatment (e.g., surgery) and who appears to be clinicallyfree of the lymphoma. The biological composition of the invention can beadministered adjunctively with any of the treatment modalities, such asbut not limited to chemotherapy, radiation, and/or surgery. For example,the biological composition can be used in combination with one or morechemotherapeutic or immunotherapeutic agents, such as vincristine,prednisone, doxorubicin, bleomycin, vinblastine, methotrexate,dexamethasone and leucovorin. The biological composition can also beused after other regimen(s) of treatment is concluded.

[0097] The subject may be one who has not yet been diagnosed withlymphoma but are predisposed to or at high risk of developing lymphomaas a result of genetic factors and/or environmental factors. The subjectmay also be one who displays characteristics that are associated with ahigh risk of lymphoma, such as nodules detected by computer tomographicscanning or suspect cells in biopsy and/or body fluids.

[0098] Depending on the subject, the therapeutic and healthful benefitsrange from inhibiting or retarding the growth of the lymphoma and/or thespread of the lymphoma to other parts of the body (i.e., metastasis),palliating the symptoms of the cancer, improving the probability ofsurvival of the subject with the cancer, prolonging the life expectancyof the subject, improving the quality of life of the subject, and/orreducing the probability of relapse after a successful course oftreatment (e.g., surgery, chemotherapy or radiation). The symptomsassociated with lymphoma include painless swelling in one or more of thelymph nodes of the neck, collarbone region, armpits, or groin., chestpain, coughing, fatigue, shortness of breath, fever, drenching nightsweats, weight loss, fatigue, appetite loss, red patches on the skin,and severely itchy skin, often affecting the legs/feet.

[0099] In particular, the invention provides a method for retarding thegrowth of lymphoma cells in a subject, such as a human, comprisingadministering orally to the subject a biological composition of theinvention. The invention also provide a method for prolonging the timeof survival of a subject inflicted with lymphoma, preferably a humanpatient, comprising administering orally to the subject a biologicalcomposition of the invention.

[0100] The effective dose will vary with the subject treated. Theeffective dose for the subject will also vary with the condition to betreated and the severity of the condition to be treated. The dose, andperhaps the dose frequency, will also vary according to the age, bodyweight, and response of the individual subject. In general, the totaldaily dose range of activated and conditioned yeast cells for a subjectinflicted with lymphoma is from about 10⁵ to 10¹¹ cells per day;preferably, about 10⁸ to 5×10¹⁰ cells per day; more preferably, about2×10⁹ cells per day in powder form or 9×10⁸ to 1×10¹⁰ cells per day inliquid preparations, administered in single or divided doses orally. Thelength of time for a course of treatment should be at least 1 week, atleast 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, atleast 7 weeks, at least 10 weeks, at least 13 weeks, at least 15 weeks,at least 20 weeks, at least 6 months, or at least 1 year. It may benecessary to use dosages outside these ranges in some cases as will beapparent to those skilled in the art. In certain embodiments, the oralcompositions can be administered for a period of time until the symptomsand/or infection of the patients by the bacteria and viruses are undercontrol, or when the disease has regressed partially or completely. Foruse as a dietary supplement, the total daily dose range should be fromabout 10⁵ to 10¹¹ cells per day; preferably, about 5×10⁷ to 5×10⁹ cellsper day. The oral compositions can be administered as a dietarysupplement for as long as 6 months, or in accordance with recommendedlength of use under the Dietary Supplement Health and Education Act(DSHEA) or other government or industry guidelines. Further, it is notedthat the nutritionist, dietician, clinician or treating physician willknow how and when to interrupt, adjust, or terminate use of thebiological composition as a medicament or dietary supplement inconjunction with individual patient response.

[0101] The effect of the biological compositions of the invention ondevelopment and progression of lymphoma can be monitored by any methodsknown to one skilled in the art, including but not limited to measuring:a) changes in the size and morphology of the tumor using imagingtechniques such as a computed tomographic (CT) scan or a sonogram; andb) changes in levels of biological markers of risk for lymphoma.

5.2.2 Formulations

[0102] The biological compositions of the present invention compriseactivated and conditioned live yeast cells prepared as described abovein Section 5. 1, as active ingredient, and can optionally contain apharmaceutically acceptable carrier or excipient, and/or otheringredients provided that these ingredients do not kill or inhibit theyeast cells. Other ingredients that can be incorporated into thebiological compositions of the present invention, may include, but arenot limited to, herbs (including traditional Chinese medicine products),herbal extracts, vitamins, amino acids, metal salts, metal chelates,coloring agents, flavor enhancers, preservatives, and the like.

[0103] Any dosage form may be employed for providing the subject with aneffective dosage of the oral composition. Dosage forms include tablets,capsules, dispersions, suspensions, solutions, and the like. In oneembodiment, compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets, or tablets, each containing a predetermined amount of activatedand conditioned yeast cells, as a powder or granules or as a solution ora suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. Such products can be used as pharmaceuticals or dietarysupplements, depending on the dosage and circumstances of its use.

[0104] The oral compositions of the present invention may additionallyinclude binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); binders orfillers (e.g., lactose, pentosan, microcrystalline cellulose or calciumhydrogen phosphate); lubricants (e.g., magnesium stearate, talc orsilica); disintegrants (e.g., potato starch or sodium starch glycolate);or wetting agents (e.g., sodium lauryl sulphate). The tablets orcapsules can be coated by methods well known in the art.

[0105] Liquid preparations for oral administration can take the form of,for example, solutions, syrups or suspensions, or they can be presentedas a dry product for constitution with water or other suitable vehiclebefore use. The temperature of the liquid used to reconstitute the driedproduct should be less than 65° C. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). As describedbelow, the preparations can also be made to resemble foods or beverages,containing buffer salts, flavoring, coloring and sweetening agents asappropriate. In certain embodiments, the oral composition is a cellsuspension comprising about 10³ to 10¹⁰ cells per ml. The oralcomposition can be produced by diluting or concentrating the yeastculture medium produced by the method of Section 5.1.5 as required. Inless preferred embodiments, the oral composition is a cell suspensioncontaining about 10³ to 10⁶ cells per ml. In more preferred embodiments,the oral composition is a cell suspension containing greater than about10⁶ to 10¹⁰ cells per ml. In most preferred embodiments, the oralcomposition is a cell suspension containing about 10⁶ to 5×10⁸ cells perml. The oral composition can be formulated as a health drink andpackaged in liquid containers, each containing a predetermined amount ofthe liquid yeast culture. Standard methods of quality control andpackaging are applied to produce in one embodiment of the invention,oral compositions packaged in liquid containers each comprising about 1ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml, 75ml, 100 ml, 150 ml, 200 ml, 250 ml, 500 ml, 750 ml, or 1,000 ml of thelive yeast cells. The number of container to be taken each day to obtainthe total daily dose in a subject depends on the number of activated andconditioned yeast cells contained within each container. For example, acontainer may comprise 50 ml of liquid with 10⁷ cells per ml and when atotal daily dose of about 2×10⁹ cells per day is desired, a subject candrink 4 containers per day to obtain the desired total daily dose.

[0106] Generally, because of their ease of administration, tablets andcapsules represent the most advantageous oral dosage unit form, in whichcase solid pharmaceutical carriers as described above are employed. In apreferred embodiment, the composition is a capsule. The capsules can beformulated by any commercially available methods. In certainembodiments, the composition is a capsule containing 5 mg, 10 mg, 15 mg,20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1.0 gram, 1.25 gram,1.5 gram, or 2.0 gram of live yeast cells in powder form. The powder inthe capsule comprises about 10⁵ to about 10¹¹ cells per gram; morepreferably, about 10⁸ to 5×10¹⁰ cells per gram; and most preferably,about 5×10⁸ cells per gram. The number of capsule to be taken each dayto obtain the total daily dose in a subject depends on the number ofactivated and conditioned yeast cells contained within each capsule. Forexample, a capsule may comprise about 500 mg of powder with 5×10⁸ cellsper gram. To achieve a total daily dose of about 2×10⁹ cells per day, asubject can take two capsules at a time for four times per day.

[0107] In another embodiment, the biological compositions comprisingactivated and conditioned yeast cells can be added directly to foods sothat an effective amount of yeast cells is ingested during normal meals.Any methods known to those skilled in the art may be used to add to orincorporate the biological compositions into natural or processed foods,provided that the activated and conditioned yeast cells remain viable.Preferably, the nutritional compositions of the invention are made andstored under conditions, such as temperature, from about 0° C. to 4° C.As used herein, the term “food” broadly refers to any kind of material,liquid or solid, that is used for nourishing an animal, and forsustaining normal or accelerated growth of an animal including humans.Many types of food products or beverages, such as but not limited to,fruit juice, herbal extracts, tea-based beverages, dairy products,soybean product (e.g., tofu), and rice products, can be used to formnutritional compositions comprising the activated and conditioned yeastcells of the invention.

[0108] The invention is further defined by reference to the followingexample describing in detail the animal trials conducted to study theefficacy and safety of activated and conditioned yeast cells of theinvention.

6. EXAMPLE

[0109] An oral composition of the invention was tested in two models oflymphoma in mouse. The oral composition was prepared using Saccharomycescerevisiae strain AS2.562 as described in Sections 5.1.3 to 5.1.5 above.The activated and conditioned yeast cells were at a concentration of1×10⁸ per ml.

6.1 Inbred PBA Mouse Model 6.1.1 Methods and Materials

[0110] A model based on inbred PBA mice which manifest a high incidenceof tumor was used (Bailey et al., 1970, J Natl. Cancer Inst.45(1):59-73). Mice six to eight weeks old were treated by the method asdescribed in Shiyan Dongwu Yu Zhongliu Yanjiu (Experimental Animal &Tumor Research), April 2000, by Chinese Medical Technology Publisher.

[0111] The mice that developed lymphoma spontaneously and that had beentreated by the method as described above were divided into two series offive groups: one series received treatment for 30 days, the other seriesreceived treatment for 60 days. In any one experimental series, eachtreatment group has a total of 36 mice.

[0112] In the first series, the mice in group AY received 0.3 ml of theoral composition of the invention per day. The mice in group NY received0.3 ml per day of a composition comprising at a similar concentrationthe same strain of yeast which had not been activated and conditioned.The mice in group CTX received subcutaneous injection ofcyclophosphamide at a dose of 30 mg per kg body weight per day. The micein group CK1 received physiological saline, while the mice in group CK2received physiological saline but did not receive transplanted lymphomacells. The experiment was carried out over a 30-day treatment period.

[0113] In the second series, the mice in group 2AY received 0.5 ml ofthe oral composition of the invention per day. The mice in group 2NYreceived 0.5 ml per day of a composition comprising the same strain ofyeast which had not been activated and conditioned at a similarconcentration. The mice in group 2CTX received subcutaneous injection ofcyclophosphamide at a dose of 45 mg per kg body weight per day. The micein group 2CK1 received physiological saline, while the mice in group2CK2 received physiogical saline but did not receive lymphoma cells. Theexperiment was carried out over a 60-day treatment period.

[0114] After the 30-day or 60-day treatment, the mice were fed a normaldiet and allowed to live under the same environment. The health andsurvival of the mice were observed and compared over 180 days.

6.1.2 Results

[0115] The results of the first series of five groups which receivedtreatment for a total of 30 days are shown in Table 4. Each groupconsisted of 36 mice at the beginning of the experiment (3 repeats). Thetotal number of surviving mice in each group were counted at the end ofevery month up to 6 months after treatment stopped. TABLE 4 Group GroupCTX Group NY Days CK2 (30 mg/kg Group AY (0.3 ml non- after (saline, nocyclophosphamide (0.3 ml oral activated treatment lymphoma Group CK1 mlper compositions yeasts per period cells) (saline only) day) per day)day) 0 36 34 36 36 33 30 36 22 33 36 26 60 36 0 27 36 21 90 36 0 19 36 0120 36 0 17 34 0 150 36 0 0 34 0 180 36 0 0 34 0

[0116] The results of the second series of five groups which receivedtreatment for a total of 60 days are shown in Table 5. Each groupconsisted of 36 mice at the beginning of the experiment (3 repeats). Thetotal number of surviving mice in each group were counted at the end ofevery month up to 6 months after treatment stopped. TABLE 5 Group GroupGroup 2NY Days 2CK2 2CTX (30 mg/kg Group 2AY (0.5 ml non- after (saline,no Group cyclophosphamide (0.5 ml oral activated treatment lymphoma 2CK1(saline ml compositions yeasts per period cells) only) per day) per day)day) 0 36 36 36 36 35 30 36 25 36 36 28 60 36 11 29 36 19 90 36 4 21 3611 120 36 0 14 36 6 150 36 0 14 36 0 180 36 0 8 36 0

[0117] The above results indicate that the lifespan of PBA mice withspontaneous lymphoma was prolonged as a result of receiving the oralcomposition of the invention at a dose of 0.3 ml per day or 0.5 ml perday for a 30-day or 60-day treatment period. The results also showedthat in comparison to cyclophosphamide, the oral composition of theinvention at a dose of 0.3 ml per day or 0.5 ml per day for a 30-day or60-day treatment period prevented the recurrence of the lymphoma in theanimals after treatment ceased.

6.2 Athymic Nude Mouse Model 6.2.1 Methods and Material

[0118] A model based on transplantation of Burkitt's lymphoma cell inathymic nude mice was used (Gurtsevitch et al., 1988, Int J Cancer15;41(1):87-95). The model was set up as described in Shiyan Dongwu YuZhongliu Yanjiu (Experimental Animal & Tumor Research), April 2000, byChinese Medical Technology Publisher. Essentially, seven week old micewere injected intravenously each with 1×10⁶ Burkitt's lymphoma cells.

[0119] Twenty-one days after the injection of lymphoma cells, the micewere divided into two series of five groups: one series receivedtreatment for 30 days, the other series received treatment for 60 days.In any one experimental series, each treatment group has a total of 36mice. The mice in group A received saline orally. The mice in group Breceived subcutaneous injection of cyclophosphamide at a dose of 30 mgper kg body weight per day. Group C received subcutaneous injection ofcyclophosphamide at a dose of 45 mg per kg body weight per day. The micein group D received 0.3 ml of the oral composition per day. Group Ereceived 0.5 ml of the oral composition per day.

[0120] After the 30-day or 60-day treatment, the mice were fed a normaldiet and allowed to live under the same environment. The health andsurvival of the nude mice were observed and compared over 360 days.

6.2.2 Results

[0121] The results of the first series of five groups which receivedtreatment for a total of 30 days are shown in Table 7. Each groupconsisted of 36 mice at the beginning of the experiment (3 repeats). Thetotal number of surviving mice in each group were counted at the end ofevery month up to 12 months after treatment stopped. TABLE 6 Group BGroup C (30 mg/kg (45 mg/kg Group D Group E Days after Group Acyclophosphamide cyclophosphamide (0.3 ml oral (0.5 ml oral treatment(0.5 saline ml ml compositions compositions period per day) per day) perday) per day) per day) 0 36 36 35 36 36 30 36 33 35  35* 36 60 36 24 2735 36 90  35* 14 21 35 36 120 35 5 17 35 36 150 35 3 11 35 36 180 35 1 435 36 210 35 0 2 35 36 240 35 0 1 35 36 270 35 0 0 35  34* 300  34* 0 035 34 330 34 0 0 35 34 360 34 0 0 35 34

[0122] The results of the second series of five groups which receivedtreatment for a total of 60 days are shown in Table 6. Each groupconsisted of 36 mice at the beginning of the experiment (3 repeats). Thetotal number of surviving mice in each group were counted at the end ofevery month up to 12 months after treatment stopped. TABLE 7 Group BGroup C (30 mg/kg (45 mg/kg Group D Group E Days after Group Acyclophosphamide cyclophosphamide (0.3 ml oral (0.5 ml oral treatment(0.5 saline ml ml compositions compositions period per day) per day) perday) per day) per day) 0 36 35 34  35* 36 30 36 35 34 35 36 60 36 33 3435 36 90 36 24 27 35 36 120 36 19 19 35 36 150 36 9 12 35 36 180 36 5 735 36 210 36 2 3 35 36 240 36 0 0 35 36 270 36 0 0 35  35* 300 36 0 0 3535 330  34* 0 0 35 35 360 34 0 0 35 35

[0123] The above results indicate that the lifespan of athymic nude micetransplanted with Burkitt's lymphoma cells was prolonged as a result ofreceiving the oral composition of the invention at a dose of 0.3 ml perday or 0.5 ml per day for a 30-day or 60-day treatment period. Theresults also showed that in comparison to cyclophosphamide, the oralcomposition of the invention at a dose of 0.3 ml per day or 0.5 ml perday for a 30-day or 60-day treatment period prevented the recurrence ofthe lymphoma in the animals after treatment ceased.

[0124] The present invention is not to be limited in scope by thespecific embodiments described which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

[0125] All publications, patents and patent applications mentioned inthis specification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

What is claimed is:
 1. A biological composition comprising activatedyeast cells, wherein said yeast cells are prepared by a methodcomprising at least two steps selected from the group consisting of: (a)culturing yeast cells in a first electromagnetic field having afrequency in the range of 7,972 to 7,982 MHz and a field strength in therange of 250 to 300 mV/cm; (b) culturing the yeast cells in a secondelectromagnetic field having a frequency in the range of 7,993 to 8,003MHz and a field strength in the range of 250 to 300 mV/cm; (c) culturingthe yeast cells in a third electromagnetic field electromagnetic fieldshaving a frequency in the range of 10,024 to 10,032 MHz and a fieldstrength in the range of 370 to 400 mV/cm; (d) culturing the yeast cellsin a fourth electromagnetic field having a frequency in the range of12,832 to 12,842 MHz and a field strength in the range of 420 to 450mV/cm; and (e) culturing the yeast cells in a fifth electromagneticfield having a frequency in the range of 12,857 to 12,867 MHz and afield strength in the range of 310 to 350 mV/cm.
 2. A biologicalcomposition comprising activated and conditioned yeast cells, whereinthe yeast cells are prepared by a method comprising activating the yeastcells, said activating comprising at least two steps selected from thegroup consisting of: (a) culturing yeast cells in a firstelectromagnetic field having a frequency in the range of 7,972 to 7,982MHz and a field strength in the range of 250 to 300 mV/cm; (b) culturingthe yeast cells in a second electromagnetic field having a frequency inthe range of 7,993 to 8,003 MHz and a field strength in the range of 250to 300 mV/cm; (c) culturing the yeast cells in a third electromagneticfield electromagnetic fields having a frequency in the range of 10,024to 10,032 MHz and a field strength in the range of 370 to 400 mV/cm; (d)culturing the yeast cells in a fourth electromagnetic field having afrequency in the range of 12,832 to 12,842 MHz and a field strength inthe range of 420 to 450 mV/cm; and (e) culturing the yeast cells in afifth electromagnetic field having a frequency in the range of 12,857 to12,867 MHz and a field strength in the range of 310 to 350 mV/cm, andconditioning the activated yeast cells, said conditioning comprising atleast one step selected from the group consisting of: (f) culturing theyeast cells in a liquid medium comprising wild hawthorn juice andgastric juice of a mammal in a sixth electromagnetic field having afrequency in the range of 12,832 to 12,842 MHz and a field strength inthe range of 380 to 400 mV/cm; and (g) culturing the yeast cells in aliquid medium comprising wild hawthorn juice and gastric juice of amammal in a seventh electromagnetic field having a frequency in therange of 12,857 to 12,867 MHz and a field strength in the range of 300to 350 mV/cm.
 3. A biological composition comprising activated andconditioned yeast cells, wherein the activated and conditioned yeastcells of claim 2 are subjected to at least one period of culturing in aliquid medium comprising wild hawthorn juice, jujube juice, wu wei zijuice, and soybean juice, and in the presence of in any order: (h) aneighth electromagnetic field or series of electromagnetic fields havinga frequency in the range of 12,832 to 12,842 MHz and a field strength inthe range of 230 to 450 mV/cm; and (i) a ninth electromagnetic field orseries of electromagnetic fields having a frequency in the range of12,857 to 12,867 MHz and a field strength in the range of 120 to 380mV/cm.
 4. The biological composition of claim 1, wherein the activatedyeast cells are cells of Saccharomyces.
 5. The biological composition ofclaim 1, wherein the activated yeast cells are cells of Saccharomycescerevisiae Hansen strain AS2.562.
 6. The biological composition of claim1, wherein the activated yeast cells are at a concentration of about 10⁶to 10⁹ cells per ml.
 7. The biological composition of claim 1, whereinthe activated yeast cells are dried and at a concentration of about 10⁷to 10¹⁰ cells per gram.
 8. The biological composition of claim 2 or 3,wherein the activated and conditioned yeast cells are cells ofSaccharomyces.
 9. The biological composition of claim 2 or 3, whereinthe activated and conditioned yeast cells are cells of Saccharomycescerevisiae Hansen strain AS2.562.
 10. The biological composition ofclaim 2 or 3, wherein the activated and conditioned yeast cells are at aconcentration of about 10⁷ to 10¹⁰ cells per ml.
 11. The biologicalcomposition of claim 2 or 3, wherein the activated and conditioned yeastcells are dried and at a concentration of about 10⁸ to 10¹¹ cells pergram.
 12. A composition comprising the activated and conditioned yeastcells of claim 2 or 3, wherein the activated and conditioned yeast cellsare packaged in a solid dosage form.
 13. The composition of claim 12,wherein the solid dosage form comprises 10⁷ to 10¹¹ yeast cells pergram.
 14. A pharmaceutical composition comprising the activated andconditioned yeast cells of claim 2 or 3, and a pharmaceutical acceptablecarrier.
 15. A dietary supplement comprising the activated andconditioned yeast cells of claim 2 or 3, and one or more ingredientsselected from the group consisting of vitamins, herbs, herbal extracts,minerals, amino acids, metal chelates, plant extracts, coloring agents,flavor enhancers and preservatives.
 16. A nutritional compositioncomprising the activated and conditioned yeast cells of claim 2 or 3,and a food product selected from the group consisting of a fruitjuice-based beverage, a tea-based beverage, a dairy product, a soybeanproduct, and a rice product.
 17. A method for preparing a biologicalcomposition comprising activated yeast cells, said method comprising atleast two steps selected from the group consisting of: (a) culturingyeast cells in a first electromagnetic field having a frequency in therange of 7,972 to 7,982 MHz and a field strength in the range of 250 to300 mV/cm; (b) culturing the yeast cells in a second electromagneticfield having a frequency in the range of 7,993 to 8,003 MHz and a fieldstrength in the range of 250 to 300 mV/cm; (c) culturing the yeast cellsin a third electromagnetic field electromagnetic fields having afrequency in the range of 10,024 to 10,032 MHz and a field strength inthe range of 370 to 400 mV/cm; (d) culturing the yeast cells in a fourthelectromagnetic field having a frequency in the range of 12,832 to12,842 MHz and a field strength in the range of 420 to 450 mV/cm; and(e) culturing the yeast cells in a fifth electromagnetic field having afrequency in the range of 12,857 to 12,867 MHz and a field strength inthe range of 310 to 350 mV/cm.
 18. A method for preparing a biologicalcomposition comprising activated and conditioned yeast cells, saidmethod comprising activating the yeast cells, said activating comprisingat least two steps selected from the group consisting of: (a) culturingyeast cells in a first electromagnetic field having a frequency in therange of 7,972 to 7,982 MHz and a field strength in the range of 250 to300 mV/cm; (b) culturing the yeast cells in a second electromagneticfield having a frequency in the range of 7,993 to 8,003 MHz and a fieldstrength in the range of 250 to 300 mV/cm; (c) culturing the yeast cellsin a third electromagnetic field electromagnetic fields having afrequency in the range of 10,024 to 10,032 MHz and a field strength inthe range of 370 to 400 mV/cm; (d) culturing the yeast cells in a fourthelectromagnetic field having a frequency in the range of 12,832 to12,842 MHz and a field strength in the range of 420 to 450 mV/cm; and(e) culturing the yeast cells in a fifth electromagnetic field having afrequency in the range of 12,857 to 12,867 MHz and a field strength inthe range of 310 to 350 mV/cm, and conditioning the activated yeastcells, said conditioning comprising at least one step selected from thegroup consisting of: (f) culturing the yeast cells in a liquid mediumcomprising wild hawthorn juice and gastric juice of a mammal in a sixthelectromagnetic field having a frequency in the range of 12,832 to12,842 MHz and a field strength in the range of 380 to 400 mV/cm; and(g) culturing the yeast cells in a liquid medium comprising wildhawthorn juice and gastric juice of a mammal in a seventhelectromagnetic field having a frequency in the range of 12,857 to12,867 MHz and a field strength in the range of 300 to 350 mV/cm.
 19. Amethod of making a biological composition comprising activated andconditioned yeast cells, said method comprising culturing the activatedand conditioned yeast cells prepared by the method of claim 18 in aliquid medium comprising wild hawthorn juice, jujube juice, wu wei zijuice, and soybean juice, and in the presence of in any order: (h) aneighth electromagnetic field or series of electromagnetic fields havinga frequency in the range of 12,832 to 12,842 MHz and a field strength inthe range of 230 to 450 mV/cm; and (i) a ninth electromagnetic field orseries of electromagnetic fields having a frequency in the range of12,857 to 12,867 MHz and a field strength in the range of 120 to 380mV/cm.
 20. The method of claim 18 or 19 further comprising after theculturing step drying the activated and conditioned yeast cells.
 21. Themethod of claim 20, wherein the drying step comprises: (a) drying at atemperature not exceeding 65° C. for a period of time such that theyeast cells become dormant; and (b) drying at a temperature notexceeding 70° C. for a period of time to reduce the moisture content tobelow 5%.
 22. A method for retarding the growth of lymphoma cells in amammal comprising administering orally to the mammal an effective amountof the biological composition of claim 2 or
 3. 23. A method forprolonging the time of survival of a mammal with lymphoma comprisingadministering orally to the mammal an effective amount of the biologicalcomposition of claim 2 or
 3. 24. The method of claim 22, wherein saidactivated and conditioned yeast cells in the biological composition areSaccharomyces cells.
 25. The method of claim 22, wherein said activatedand conditioned yeast cells in the biological composition areSaccharomyces cerevisiae Hansen strain AS2.562.
 26. The method of claim22, wherein said activated and conditioned yeast cells in the biologicalcomposition are at a concentration of about 10⁸ cells per ml.
 27. Themethod of claim 23, wherein said activated and conditioned yeast cellsin the biological composition are Saccharomyces cells.
 28. The method ofclaim 23, wherein said activated and conditioned yeast cells in thebiological composition are Saccharomyces cerevisiae Hansen strainAS2.562.
 29. The method of claim 23, wherein said activated andconditioned yeast cells in the biological composition are at aconcentration of about 10⁸ cells per ml.